Historically, natural products have made many significant accomplishments in the development of anticancer chemotherapeutics. However, to continue our battle against aggressive cancers with low survival rates, in par- ticular metastatic and drug resistant cancers, it is crucial to explore new families of cytotoxic natural products with novel structures and/or mechanisms. Ipomoeassins are a family of plant-derived macrolides with embedded carbohydrates. Because of the potent cellular activity and distinct activity profile revealed in the 60-cell lines screen, the ipomoeassins had been considered for anti-cancer drug development by the NCI. However, this plan was hampered due to low material availability from nature. In the current funding period, we have made signifi- cant progress in the field. Using the most potent member, ipomoeassin F (Ipom-F), as the target molecule, we first developed its gram-scale total synthesis. Subsequently, we performed the most sophisticated medicinal chemistry studies for design and syntheses of novel functional probes of Ipom-F, with which we identified and confirmed Sec61? (protein transport protein Sec61 subunit alpha isoform 1) as the primary protein target of Ipom-F. Therefore, Ipom-F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61? function and to further investigate its potential as a thera- peutic target for drug discovery. Following these results, the overall objective of this renewal proposal is to use simplified open-chain mimics of Ipom-F to collect structural information on how Ipom-F binds Sec61? at the atomic level, which is missing knowledge in Sec61 research. Our rationale for the proposed studies is that the information and materials that can be enriched more efficiently with open-chain analogues will considerably fa- cilitate future biomedical evaluations of new pharmaceutical agents derived from this unique class of natural products. Our hypothesis is that the dynamic ?open-state? lateral gate channel of Sec61? can effectively fold open-chain analogues of Ipom-F into the ?closed-state? of Sec61? with very little entropy penalty. A set of inno- vative studies will be conducted to test this hypothesis: (1) Further structural and mechanistic investigation of Ipom-F by using its photo-crosslinking open-chain probes in conjunction with proteomics techniques; (2) Syn- thesis of two libraries of peptide-containing open-chain analogues of Ipom-F by using a novel ?amino acid scan- ning? approach, followed by efficacy evaluation both in vitro and in live cells. All the planned studies are significant because they represent a substantial departure of ipomoeassin research from its status quo. We anticipate that open-chain analogues/probes of Ipom-F will provide more accessible new molecular tools that will help on-going efforts to fully define the molecular basis for Sec61-mediated protein translocation at the ER membrane and to develop the next generation of Sec61? binders. From the educational perspective, such a synthetic project fits undergraduate students? schedule better. Interdisciplinary nature of the project will attract the students to receive training in health-related research rooted in basic science.